金纳米材料在肿瘤治疗中的应用及其对细胞自噬的调控

发布时间：2018-05-18 07:26

本文选题：金纳米材料 + 肿瘤治疗　；参考：《东南大学》2017年博士论文

【摘要】：金纳米颗粒是一种非常重要的金属纳米材料,其独特的光学性质,易控的表面化学性能和良好的生物相容性使其成为基础研究及应用研究的热点。由于金具有较高的原子序数,可以增强放射线对肿瘤区域的影响,因而金纳米材料被广泛地用于肿瘤放疗增敏。某些金纳米材料(如金纳米棒和金纳米刺)的近红外吸收特性使其具有独特的光热效应,从而被广泛地用于肿瘤的联合治疗。另外,放射治疗还可以诱导细胞自噬应激反应,并且自噬与肿瘤存在双重关系,这一特性也为肿瘤防治提供了两种截然不同的思路:抑制自噬提高抗癌治疗效果,或激活自噬诱导肿瘤细胞发生自噬性死亡。因此,目前尚不能盲目地将自噬诱导剂或抑制剂应用于临床,否则不但不能阻止肿瘤的发生,反而会促进肿瘤的发展。基于以上研究背景,本文将以表面粗糙且呈刺突状的金纳米刺(GNSs)为研究主体,围绕金纳米刺在肿瘤放疗和热疗中的应用做了一系列研究。具体说来,主要包含以下几方面的内容:1.比较研究不同形貌的金纳米材料的放疗增敏特性为了深入阐述不同形貌的金纳米材料对放射治疗的影响,我们选用了粒径相同(50 nm左右)且表面均为聚乙二醇(PEG)修饰的三种金纳米结构,即金纳米球、金纳米刺、金纳米棒,并对其放疗增敏特性进行了系统性地评价。实验发现,金纳米球、金纳米刺、金纳米棒的放疗增敏比(SER)依次为1.62、1.37、1.21,表明三种金纳米结构均具有放疗增敏效应,且材料的SER与细胞摄取率之间存在正相关性。通过进一步对SER与细胞摄取率的比值进行归一化分析,结果发现,平均每个金原子对放疗增敏的贡献几乎一致。也就是说,为实现更大程度的癌细胞杀伤效率,只需要增加金原子的细胞摄取效率即可。通过设计特定形貌的金纳米结构可实现最大限度的细胞内化。该项研究对于如何设计有效的金纳米结构以用于癌症的放射治疗具有重要的指导意义。2.配体修饰的金纳米刺放疗增敏作用及细胞自噬应激反应由于金纳米刺(GNSs)细胞摄取率低,极大地限制了其放疗增敏效果。为此我们制备得到了表面均为PEG修饰且末端带有不同配体(包括甲氧基、氨基NH2、叶酸FA和细胞穿膜肽TAT)的金纳米刺(即GNSs、NH2-GNSs、FA-GNSs和TAT-GNSs),并评价了它们在细胞摄取率和放疗增敏效果方面的差异。实验发现,不同配体修饰的金纳米刺的细胞摄取率依次为:TAT-GNSs FA-GNSs NH2-GNSs GNSs。这些材料的放疗增敏比SER(依次为2.30、1.84、1.57和1.34)与其细胞摄取率相一致:即TAT-GNSs FA-GNSs NH2-GNSsGNSs。相比于单一 PEG化的金纳米刺(端基为甲氧基的GNSs),配体修饰的金纳米刺可显著提高癌细胞的放疗增敏效应。我们还进一步从材料的细胞摄取路径、活性氧产生、线粒体膜电位变化、细胞周期和凋亡等生物学效应角度,深入阐述了放疗增敏的潜在机制。结果表明,配体修饰的金纳米刺联合放疗可诱导细胞内活性氧的产生、线粒体膜电位的下降和细胞周期的再分布等。其中,活性氧增多在其中起关键作用。此外,我们还探索了细胞自噬与放疗增敏之间的关系。结果显示,自噬作为一种溶酶体依赖的降解途径,在金纳米刺介导的放射治疗中扮演重要角色。蛋白印迹检测结果表明,配体修饰的金纳米刺能够诱导自噬相关蛋白(LC3-Ⅱ)和凋亡相关蛋白(caspase-3)的表达增多;通过对自噬降解底物p62蛋白的定量分析,我们发现金纳米刺可诱导p62的表达增多,这表明金纳米刺可损害自噬溶酶体降解能力并导致自噬小体的积累。在自噬抑制剂3-MA的作用下,PEG化的金纳米刺及其联合放射治疗均使细胞凋亡比率明显增加,即自噬在放疗中起保护性作用,而抑制自噬则可极大地促进细胞凋亡。该研究加深了我们对金纳米材料细胞摄取规律的认识,并明确了自噬抑制剂可以显著增加放疗抗癌效果。3.金纳米刺介导的高热效应在光热和放疗协同抗癌中的应用基于金纳米刺良好的光热效应和放疗增敏特性,我们建立了一种基于金纳米刺的光热/放疗联合抗癌策略。通过大量的体外和体内实验,系统性地评价了金纳米刺介导的光热/放射联合治疗效果。金纳米刺的SER为1.38,而金纳米刺联合光热治疗的SER增加到1.63,这表明金纳米刺介导的高热效应显著增强了放射线对乏氧癌细胞的杀伤效应。此外,体外实验发现金纳米刺介导的光热治疗、放射治疗以及联合治疗的癌细胞存活率分别为89%、51%和33%。体内实验显示金纳米刺介导的放射治疗、光热治疗以及联合治疗对肿瘤增殖的抑制率分别为29.8%、70.5%和92.2%。该项研究结果表明,金纳米刺介导的热疗和放疗具有优异的协同抗癌效果。该研究为多功能金纳米材料的开发和放射治疗提供了新的思路。
[Abstract]:Gold nanoparticles are one of the most important metal nanomaterials. Their unique optical properties, easy to control surface chemical properties and good biocompatibility make it a hot spot in basic research and application research. Because of the high atomic number of the metal tools, it can enhance the effect of radiation on the tumor area. Therefore, gold nanoparticles are widely used. The near infrared absorption properties of some gold nanomaterials (such as gold nanorods and gold nanoscale spines) have unique photothermal effects and are widely used in combined treatment of tumors. In addition, radiation therapy can also induce autophagy to induce autophagy and the dual relationship between autophagy and tumor. Two different ideas are provided for the prevention and treatment of tumor: inhibiting autophagy to improve the effect of anticancer therapy, or activating autophagy to induce autophagic cells to occur autophagic death. Therefore, it is not possible to apply autophagy inducers or inhibitors to the clinic blindly. Otherwise, it can not only prevent the occurrence of tumor, but also promote the development of tumor. In this research background, this paper will take a series of studies on the application of gold nano spines (GNSs) with rough surface and stings as the main body. A series of studies have been done around the application of gold nano spines in tumor radiotherapy and hyperthermia. In particular, the main contents are as follows: 1. the radiation sensitization characteristics of gold nanoparticles with different morphologies are compared. The effects of gold nanomaterials with different morphologies were discussed. We selected three gold nanostructures with the same particle size (about 50 nm) and the surface of the gold nanoparticles modified by polyethylene glycol (PEG), namely gold nanoparticles, gold nanoscale and gold nanorods. The radiosensitization ratio (SER) of the gold nanorods is 1.62,1.37,1.21 in turn, indicating that the three gold nanostructures have the effect of radiation sensitization and there is a positive correlation between the material's SER and the cell uptake. The result of the further analysis of the ratio of SER to the cell uptake results shows that the average gold atom is the tribute to the radiation sensitization. The contribution is almost identical. That is to say, in order to achieve greater cancer cell killing efficiency, it only needs to increase the cell uptake efficiency of gold atoms. The maximum cell internalization can be achieved through the design of a gold nanostructure with specific morphologies. This study is heavy on how to design an effective nanoscale structure for cancer radiation therapy. The guiding significance of.2. ligand modified gold nanoscale radiosensitization and cellular autophagy response due to the low uptake rate of gold nanoparticle (GNSs) cells greatly restricts the effect of radiation sensitization. Therefore, we have prepared PEG modification on the surface and the end with different complexes (including methoxy, amino NH2, folic acid FA and cell wear. Gold nanoscale spines (GNSs, NH2-GNSs, FA-GNSs and TAT-GNSs) of membrane peptide TAT, and their differences in cell uptake and radiation sensitization were evaluated. The experimental results showed that the uptake rate of gold nanoparticles modified by different ligands was, in turn, TAT-GNSs FA-GNSs NH2-GNSs GNSs. (TAT-GNSs FA-GNSs NH2-GNSs GNSs.) and SER (2.30,1.84,1.57). And 1.34) in accordance with its cell uptake rate: TAT-GNSs FA-GNSs NH2-GNSsGNSs. is compared to a single PEG gold nanoparticle (terminal methoxy GNSs), and ligand modified gold nanoparticle can significantly increase the radiation sensitization effect of cancer cells. We further from the cell uptake pathway, reactive oxygen generation, and mitochondrial membrane potential change The potential mechanisms of cell cycle and apoptosis, such as cell cycle and apoptosis, are discussed in detail. The results show that ligand modified gold nanoparticle combined with radiotherapy can induce intracellular reactive oxygen species, the decrease of mitochondrial membrane potential and the redistribution of cell cycle. Among them, the increase of reactive oxygen species plays a key role. The relationship between autophagy and radiation sensitization is also explored. The results show that autophagy, as a lysosome dependent degradation pathway, plays an important role in gold nanoparticle mediated radiation therapy. The results of Western blot detection showed that ligand modified gold nanoparticles could induce LC3- II and apoptosis related proteins (CASPA The expression of se-3 is increased. By quantitative analysis of the autophagic degradation substrate p62 protein, we found that gold nanoparticles can induce the increase in the expression of p62, which indicates that gold nanoparticles can damage the degradation ability of autophagosomes and lead to the accumulation of autophagic bodies. Under the action of autophagic inhibitor 3-MA, the PEG nanoscale prickle and its combined radiation therapy are all made The rate of apoptosis is obviously increased, that is, autophagy plays a protective role in radiotherapy, while inhibition of autophagy can greatly promote cell apoptosis. This study deepened our understanding of the uptake of gold nanomaterials. It is clear that autophagy inhibitors can significantly increase the anticancer effect of radiotherapy.3. gold nanoparticle mediated hyperthermia effect in light and heat. Based on the good photothermal effect and radiation sensitization of gold nanoparticles, we established a combination of photothermal / radiotherapy based on gold nanoparticle. Through a large number of in vitro and in vivo experiments, we systematically evaluated the effect of the combined photothermal / radiation therapy on gold nanoparticles. SER For 1.38, the SER of gold nano spines combined with photothermal treatment increased to 1.63, which showed that the high heat effect mediated by gold nano spines significantly enhanced the killing effect of radiation on hypoxic cancer cells. In addition, in vitro, the survival rate of gold nanoparticles mediated photothermal therapy, radiation therapy and combined treatment were 89%, 51% and 33%., respectively. The internal experiment showed that gold nanoparticle mediated radiation therapy, photothermal therapy and combined treatment of tumor proliferation inhibition rate were 29.8%, 70.5% and 92.2%., respectively. The results showed that gold nanoparticle mediated thermotherapy and radiotherapy have excellent synergistic anticancer effects. This study provides the development of multi-functional gold nanomaterials and radiation therapy. A new idea.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.5;TB383.1

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